Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 6-9-2020 Designed and Developed Delivery Systems Containing Extracted Astaxanthin from Crawfish, Procambarus clarkii, Using a Novel Combined Ethanol Flaxseed Oil Ultrasound Assisted Closed Extraction System and Its Anticancer Activity in Vitro Ronson Renard Scott Sr Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Scott, Ronson Renard Sr, "Designed and Developed Delivery Systems Containing Extracted Astaxanthin from Crawfish, Procambarus clarkii, Using a Novel Combined Ethanol Flaxseed Oil Ultrasound Assisted Closed Extraction System and Its Anticancer Activity in Vitro" (2020). LSU Doctoral Dissertations. 5296. https://digitalcommons.lsu.edu/gradschool_dissertations/5296 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. DESIGNED AND DEVELOPED DELIVERY SYSTEMS CONTAINING EXTRACTED ASTAXANTHIN FROM CRAWFISH, PROCAMBARUS CLARKII, USING A NOVEL COMBINED ETHANOL FLAXSEED OIL ULTRASOUND ASSISTED CLOSED EXTRACTION SYSTEM AND ITS ANTICANCER ACTIVITY IN VITRO A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The School of Nutrition and Food Sciences by Ronson Renard Scott Sr. B.S., Louisiana State University, 2008 M.S., Louisiana State University, 2013 August 2020 © 2020/copyright Ronson Renard Scott Sr. ii First and foremost, I want to give thanks to God for his grace and mercy in allowing me to accomplish my endeavors. This study is wholeheartedly dedicated to my beloved mother, who never gave up on me throughout the tumultuous challenge and gave me strength to keep going when I was knocked down, who continued to provide spiritual, emotional, and moral support. To my siblings, relatives, friends, classmates, mentors, and major advisor who shared their words of advice and encouragement to finish my study. Also, to my life partner for her continued support throughout this process. iii ACKNOWLEDGEMENTS I would like to thank my major advisor, Dr. Subramaniam Sathivel, who guided me in this process and afforded me the opportunity to achieve my goal. His continued words of encouragement and affirmation pushed me to excel. Additionally, I would like to thank all members of my committee, Dr. Joseph Bankston, Dr. Joan King, Dr. Michael Keenan, Dr. Elizabeth Martin, and Dr. Suzanne Marchand for the time, support, and patience. Appreciation is extended to Dr. John Finley, for his encouragement to pursue my doctoral degree. I am grateful to everyone with Bocage Crawfish, LLC for their cooperative efforts and supplying me with crawfish. I would also like to thank TIC Gums for providing me with polymers. My gratitude goes to my laboratory mates at LSU, both past and present, for their comradery. I would like to thank my mother, Wanda Scott, father, Greg Scott, and everyone else that supported me emotionally. I appreciate all of my physicians that have treated me throughout the past years in an effort to improve my quality of life. I cannot forget my cousin, Clarke, as it was my promise to her years ago that this moment would come to fruition. Most notably, I want to acknowledge my Karla Lynn Huxen, for her support. Despite her busy life, she made me a priority and I am forever indebted to her. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS………………………………………………………………………iv LIST OF TABLES……………….……………………………………….…………………...…vii LIST OF FIGURES…………………………………………………………………………......viii LIST OF ABBREVIATIONS AND ACRONYMS ..……………………………………………xi ABSTRACT……………………………………………………………………………….…......xv CHAPTER 1. LITERATURE REVIEW 1.1. Louisiana seafood……….…………………………………...……………….1 1.2. Antioxidants……………………….……………………...……………..……2 1.3. Xanthophyll carotenoids: astaxanthin………………………….…………..…7 1.4. Lipid digestion and absorption………………………………………………19 1.5. Flaxseed oil…….………………………...………………………….………23 1.6. Lipid peroxidation…...………………………………………………………25 1.7. Hydrocolloids……….……………………...……………………..…………29 1.8. Cancer trends and treatment……………...……….…………………………38 CHAPTER 2. EXTRACTION OF AND CRAWFISH ASTAXANTHIN WITH FLAXSEED OIL: EFFECTS ON LIPID OXIDATION AND ASTAXANTHIN…...………..45 2.1. Introduction………………………………………………………………… 45 2.2. Materials and methods……………………………………………………... 48 2.3. Results and discussion………………………………………………………64 2.4. Conclusion…………………………………………………………………..82 CHAPTER 3. DEVELOPING ENTRAPPED FLAXSEED OIL CONTAINING CRAWFISH ASTAXANTHIN IN A PECTIN-GELATIN MATRIX GUMMY………….......84 3.1. Introduction………………………………………………………………….84 3.2. Materials and methods………………………………………………………86 3.3. Results and discussion………………………………………………………96 3.4. Conclusion…………………………………………………………………109 CHAPTER 4. ASTAXANTHIN: CHEMOTHERAPEUTIC PROPERTIES………………….111 4.1. Introduction………………………………………………………………...111 4.2. Materials and methods……………………………………………………..112 4.3. Results and discussion…………………………………………………..…115 4.4. Conclusion…………………………………………………………………120 CHAPTER 5. CONCLUSIONS…………………………………..……………………………122 REFERENCES…………………………………………………………………………………123 v VITA…………………………………………………………………………………….……...156 vi LIST OF TABLES Table 1.1. Sources of astaxanthin ……………………………………………………………12-13 Table 1.2. Fatty acid content of flaxseed oil…………………………………….……………….25 Table 1.3. Percentile of the projected (1,762,450) new cancer cases and survival rate …......38-39 Table 2.1. Proximate composition and AX content of CB, SB, freeze-dried CB, and freeze-dried SB………………………………………………………………………………….….68 Table 2.2. Physicochemical properties of FO, FOCAX, FOSAX, and FOCAXUAE…….…..…69 Table 2.3. AC, PV and TBARS of FO, FOCAX, FOSAX, 10UAE, 25UAE and 50UAE with FOAX, and NFO and CFO ..………………………………………………...……….69 Table 2.4. Color in terms of L*, a*, b*, Chroma, Hue, and color difference of FOCAX, FOSAX, HFO and FO ..…………………………...………………………………………...….72 Table 2.5. Slope, y intercept, and correlation coefficients for linear regression of POV of Oils not heated and heated at 65 ºC vs. time……………………………………………………………...78 Table 3.1. Phospholipid content in sunflower-lecithin …………………………….……………87 Table 3.2. Formulations of pectin -gelatin gummy pieces……………………………………….90 Table 3.3. Compositional parameters of pectin gelatin delivery systems (PGDS)………………97 vii LIST OF FIGURES Figure 1.1. Balance of redox equilibrium ……………...……………………………………..…..4 Figure 1.2. Carotenoid biosynthesis from lycopene………………………………………………9 Figure 1.3. Chemical structure of astaxanthin…….……………………………………………..10 Figure 1.4. Structures of optical isomers all-E-astaxanthin……………………………………...10 Figure 1.5. Geometrical isomers all-E-, 9Z-, 13Z- and 15Z-astaxanthin...……………………...11 Figure 1.6. Synthetic astaxanthin stereoisomers…………………………………………………14 Figure 1.7. Gastrointestinal system anatomy…………………………………………………….20 Figure 1.8. Lipid oxidation occurring through both enzymatic and non-enzymatic reactions…..26 Figure 1.9. Chemical structure of gelatin………………………………………………………...31 Figure 1.10. Chemical structure of hydroxypropyl methylcellulose ………………...……..…...32 Figure 1.11. Chemical structure of pectin. Alpha 1, 4 D-galacturonic acid……………………..33 Figure 1.12. Main trends in the behavior of biopolymer mixtures………………………………34 Figure 1.13. (A) MDA-MB-231 epithelial cells expressing mesenchymal-like phenotypes (high number after 2 days incubation in DMEM at 37 ºC. (B) MDA-MB-231 (ATCC® HTB-26™) epithelial cells expressing mesenchymal-like phenotypes……....……41 Figure 2.1. Vats GOM shrimp at processing facility ................................................................... 49 Figure 2.2. Shrimp being emptied onto conveyor for removal of rocks and debris before peeling process…….................................................................................................................49 Figure 2.3. Thawing of IQF shrimp .............................................................................................. 49 Figure 2.4. Crawfish farm in Crowley, LA……………...………………………….....................49 Figure 2.5. Peeling and sizing of crawfish tails………………………………………………….49 Figure 2.6. Frozen crawfish processing byproducts (peelings) .................................................... 49 Figure 2.7. Conventional extraction of astaxanthin from crustacean byproducts using flaxseed oil. FOAX = Flaxseed oil containing astaxanthin. ..................................................... 53 viii Figure 2.8. Shrimp processing byproducts ................................................................................... 53 Figure 2.9. Phase separation using separatory funnel ................................................................... 53 Figure 2.10. Centrifuge bottle containing flaxseed oil with astaxanthin………………………...54 Figure 2.11. Freeze dried crawfish processing byproducts………………………………………54 Figure 2.12. Ultrasound assisted extraction system used to extract astaxanthin. ......................... 55 Figure 2.13. Calibration curve using ascorbic acid………………………………………………63 Figure 2.14. L* value, measurement of lightness to darkness in terms of CIELAB color scale. 74 Figure 2.15. a* value, measurement of red to green in terms of CIELAB color scale. ................ 74 Figure 2.16. b* value, measurement